Applying real-time monitoring of circadian oscillations in adult mouse brain slices to study communications between brain regions.

This protocol combines a protective cutting method to prepare various brain slices from adult mice and real-time monitoring of circadian oscillations to measure circadian rhythmicity in various brain slices. This protocol can be applied to studies of how brain damages affect local circadian clocks and subsequent circadian variations in nearby areas. Further functional analyses with in vivo systems will determine whether these circadian variations are detrimental or beneficial to the brain. For complete details on the use and execution of this protocol, please refer to Huang et al. (2020).

Absolute Quantitation of Proteins by Coulometric Mass Spectrometry.

Accurate quantification is essential in the fields of proteomics, clinical assay, and biomarker discovery. Popular methods for absolute protein quantitation by mass spectrometry (MS) involve the digestion of target protein and employ isotope-labeled peptide internal standards to quantify chosen surrogate peptides. Although these methods have gained success, syntheses of isotope-labeled peptides are time-consuming and costly. To eliminate the need for using standards or calibration curves, herein we present a coulometric mass spectrometric (CMS) approach for absolute protein quantitation, based on the electrochemical oxidation of a surrogate peptide combined with mass spectrometric measurement of the oxidation yield. To demonstrate the utility of this method, several proteins were analyzed such as model proteins ß-casein, and apomyoglobin as well as circadian clock protein KaiB isolated from Escherichia coli. In our experiment, tyrosine-containing peptides were selected as surrogate peptides for quantitation, considering the oxidizable nature of tyrosine. Our data showed that the results for surrogate peptide quantity measured by our method and by traditional isotope dilution method are in excellent agreement, with the discrepancy of 0.3-3%, validating our CMS method for absolute quantitation. Furthermore, therapeutic monoclonal antibody (mAb) could be quantified by our method as well. Due to the high specificity and sensitivity of MS and no need to use isotope-labeled peptide standards, our CMS method would be of high value for the absolute proteomic quantification.

Characterization of the circadian oscillator in the choroid plexus of rats.

Circadian rhythms are a fundamental biological phenomena that control various physiological functions. The suprachiasmatic nucleus (SCN) is a master clock that integrates various peripheral clocks. Recently, the choroid plexus (CP) was reported to be one such peripheral clock, a circadian oscillator that might conversely affect the SCN. Hence, the principle aim of our study was to unravel the circadian oscillator within the CP. Quantitative PCR against rPer1, rPer2, and rBmal1 showed that CP in the lateral ventricle (CP-LV) and fourth ventricle (CP-4V) has a robust circadian oscillator. The phases of the CP oscillator are between those of the pineal gland (PG) and SCN. Bioluminescence monitoring of explants showed that the intrinsic circadian period of CP-LV and CP-4V was approximately 21 h, which is shorter than SCN and PG. It is possible that interaction between oscillators of the CP-LV, CP-4V, PG, and SCN ensures the SCN adopts a stable 24 h rhythm, with each of the regions having an intrinsic oscillator with different phases and periods. In situ hybridization analysis revealed that dusk-to-dawn variation of rPer2 expression was found in epithelial cells of the CP only. Furthermore, the CP circadian oscillator might control cerebrospinal fluid secretion. However, no dusk-to-dawn variation in expression of the water channel, aquaporin 1, was observed. Further investigations are needed to clarify the involvement of circadian rhythm on CP.

Circadian clock gene Per2 plays an important role in cell proliferation, apoptosis and cell cycle progression in human oral squamous cell carcinoma.

Previous studies have shown that the aberrant expression of period circadian clock 2 (Per2) is closely related to the occurrence and development of cancers, but the specific mechanism remains unclear. In the present study, we used shRNA to downregulate Per2 in oral squamous cell carcinoma (OSCC) Tca8113 cells, and then detected the alterations in cell cycle, cell proliferation and apoptosis by flow cytometric analysis and mRNA expression alterations in all the important genes in the cyclin/cyclin-dependent protein kinase (CDK)/cyclin-dependent kinase inhibitor (CKI) cell cycle network by RT-qPCR. We found that in the Tca8113 cells, after Per2 downregulation, the mRNA expression levels of cyclin A2, B1 and D1, CDK4, CDK6 and E2F1 were significantly increased (P<0.05), the mRNA expression levels of p53, p16 and p21 were significantly decreased (P<0.05), cell proliferation was significantly higher (P<0.05), apoptosis was significantly lower (P<0.05) and the number of cells in the G1/G0 phase was significantly decreased (P<0.05). The present study proves that in OSCC, clock gene Per2 plays an important role in cell cycle progression and the balance of cell proliferation and apoptosis by regulation of the cyclin/CDK/CKI cell cycle network. Further research on Per2 may provide a new effective molecular target for cancer treatments.

Chronic Alcohol Consumption in Rats Leads to Desynchrony in Diurnal Rhythms and Molecular Clocks.

BACKGROUND: Circadian rhythms are essential for adapting to the environment. Chronic alcohol consumption often leads to sleep and circadian disruptions, which may impair the life quality of individuals with alcohol use disorders and contribute to the morbidity associated with alcoholism. METHODS: We used a pair-feeding liquid diet alcohol exposure protocol (6 weeks duration) in PER1::LUC transgenic rats to examine the effects of chronic alcohol exposure on: (i) diurnal rhythms of core body temperature and locomotor activity, (ii) plasma corticosterone (CORT) concentrations, and (iii) rhythms of ex vivo Period1 (Per1) expression in the suprachiasmatic nucleus (SCN), pituitary, and adrenal glands. We followed multiple circadian outputs not only to examine individual components, but also to assess the relative phase relationships among rhythms. RESULTS: We found that chronic alcohol consumption: (i) reduced 24-hour body temperature and locomotor activity counts in the dark period, (ii) advanced the acrophase of diurnal rhythms of body temperature and locomotor activity, (iii) abolished the phase difference between temperature and activity rhythms, (iv) blunted and advanced the diurnal CORT rhythm, and (v) advanced Per1 expression in the adrenal and pituitary glands but not in the SCN. We found that chronic alcohol altered the phase relationships among diurnal rhythms and between the central (SCN) and peripheral (adrenal and pituitary) molecular clocks. CONCLUSIONS: Our findings suggest that desynchrony among internal rhythms is an important and overlooked aspect of alcohol-induced circadian disruptions. The misalignment of phases among rhythms may compromise normal physiological functions and put individuals with chronic alcohol use at greater risk for developing other physical and mental health issues. How this desynchrony occurs and the extent to which it participates in alcohol-related pathologies requires further investigation.

[Role of circadian rhythms and cellular clock in aging-related diseases].

Almost all the functions of a living organism have circadian oscillations. Disturbance of circadian rhythms can be either a reason or a consequence of a number of diseases. The article describes data on relationship of circadian rhythm and clock genes disturbances with type 2 diabetes mellitus, cardiovascular and neurodegenerative disease development in humans and animals.

Period 1 and estrogen receptor-beta are downregulated in Chinese colon cancers.

To investigate whether Period 1 (PER1) and Estrogen receptor-beta (ER2) are associated with occurrence and development of Chinese colorectal cancers. By using RT-quantitative PCR, tissue microarray (TMA) and immunohistochemistry, we detected mRNA levels and protein levels of PER1 and ER2 in the cancerous tissues and paired normal adjacent tissues in patients with colorectal cancer. Survival analyses were performed by the Kaplan-Meier method utilizing log-rank test and univariate and multivariate Cox proportional modeling to measure 5-year disease-free survival (DFS) and overall survival (OS). Real-time PCR showed that, the delta Ct value (tumor tissue vs. normal mucosa) of PER1 or ER2 is 8.51 ± 2.81 vs. 7.34 ± 2.08 or 12.39 ± 2.43 vs. 9.76 ± 1.75, expression of PER1 and ER2 decreased significantly in tumor tissues compared with noncancerous mucosas of patients with or without metastasis (both of P values <0.001). Spearman test revealed that PER1 and ER2 were significantly down-regulated in cancerous tissues (r=0.283; P<0.001) which was also confirmed by immunohistochemistry of specimens from 203 colon cancer patients in a TMA format. The reduction of PER1 was associated with gender and distant metastasis (P=0.037 and P<0.001, respectively) whereas the decline of ER2 was associated with age (P=0.043) by analyzing the clinical data. However, we were not capable of detecting any association between PER1 level or ER2 level and overall survival (OS) or disease free survival (DFS). It is the first observation of correlated reduction of PER1 and ER2 in Chinese colon cancers, and they do play a certain role in colorectal cancer.

Prognostic relevance of Period1 (Per1) and Period2 (Per2) expression in human gastric cancer.

Period1 (Per1) and Period2 (Per2) are members of the circadian genes. Mounting evidence suggests that the deregulation of the circadian clock plays an important role in the development of mammalian cancer. However, the expression and clinical significance of Per1 and Per2 in gastric cancer is still unexplored. Here, we evaluated the expression pattern of Per1 and Per2 in 246 gastric cancer specimens and their adjacent, non-tumorous tissues using immunohistochemical assays. Per1 expression was significantly associated with clinical stage (p < 0.001), depth invasion (p < 0.001), lymph node metastasis (p < 0.001) and pathologic differentiation (p < 0.001). On the other hand, Per2 was associated with clinical stage (p = 0.021) and depth invasion (p = 0.007). Per1 expression was positively correlated with Per2 expression in the 246 gastric cancer patients (r = 0.378, p < 0.001), and the expression levels of Per1 and Per2 were down-regulated in gastric cancer tissues when compared with adjacent, non-tumorous tissues in 45 gastric cancer samples (p < 0.001, p = 0.003). Patients with lower Per1 and Per2 tumor expression had a shorter survival time than those with higher expression. Univariate and Multivariate analyses indicated that Per2 expression is an independent prognostic factor (p = 0.023). Our results demonstrate that Per1 and Per2 may play important roles in tumor development, invasion and prognosis, and Per2 may serve as a novel prognostic biomarker of human gastric cancer.

Correlation between deregulated expression of PER2 gene and degree of glioma malignancy.

AIMS AND BACKGROUND: Growing evidence indicates that disruption of circadian rhythms may be a risk factor for the development of glioma. However, the molecular mechanisms underlying the genetic regulation of circadian rhythms in glioma cells have yet to be explored. METHODS AND STUDY DESIGN: Using immunohistochemical staining and methylation-specific PCR techniques, we examined the expression of the period 2 (PER2) gene, one of the most important clock genes, epidermal growth factor receptor (EGFR), and proliferating cell nuclear antigen (PCNA) in 92 gliomas. RESULTS: Our results revealed disturbances in the expression of PER2 in most (52.17%) glioma cells compared with the expression in nearby noncancerous cells, and indicated that PER2 gene deregulation most likely occurs via methylation of PER2 promoters. The protein expression of PCNA and EGFR was significantly higher in high-grade than low-grade gliomas (P < 0.05). Furthermore, a negative correlation was detected between the protein expression of PER2 and PCNA and EGFR in glioma. CONCLUSIONS: Because the circadian clock regulates the expression of cell cycle-related genes, we suggest that disturbances in PER2 gene expression may disrupt the regulation of the circadian clock, thus enhancing the survival of cancer cells and promoting carcinogenesis.

Clock genes show circadian rhythms in salivary glands.

Circadian rhythms are endogenous self-sustained oscillations with 24-hour periods that regulate diverse physiological and metabolic processes through complex gene regulation by "clock" transcription factors. The oral cavity is bathed by saliva, and its amount and content are modified within regular daily intervals. The clock mechanisms that control salivary production remain unclear. Our objective was to evaluate the expression and periodicity of clock genes in salivary glands. Real-time quantitative RT-PCR, in situ hybridization, and immunohistochemistry were performed to show circadian mRNA and protein expression and localization of key clock genes (Bmal1, Clock, Per1, and Per2), ion and aqua channel genes (Ae2a, Car2, and Aqp5), and salivary gland markers. Clock gene mRNAs and clock proteins were found differentially expressed in the serous acini and duct cells of all major salivary glands. The expression levels of clock genes and Aqp5 showed regular oscillatory patterns under both light/dark and complete-dark conditions. Bmla1 overexpression resulted in increased Aqp5 expression levels. Analysis of our data suggests that salivary glands have a peripheral clock mechanism that functions both in normal light/dark conditions and in the absence of light. This finding may increase our understanding of the control mechanisms of salivary content and flow.

Regulation of core clock genes in human islets.

Nearly all mammalian cells express a set of genes known as clock genes. These regulate the circadian rhythm of cellular processes by means of negative and positive autoregulatory feedback loops of transcription and translation. Recent genomewide association studies have demonstrated an association between a polymorphism near the circadian clock gene CRY2 and elevated fasting glucose. To determine whether clock genes could play a pathogenetic role in the disease, we examined messenger RNA (mRNA) expression of core clock genes in human islets from donors with or without type 2 diabetes mellitus. Microarray and quantitative real-time polymerase chain reaction analyses were used to assess expression of the core clock genes CLOCK, BMAL-1, PER1 to 3, and CRY1 and 2 in human islets. Insulin secretion and insulin content in human islets were measured by radioimmunoassay. The mRNA levels of PER2, PER3, and CRY2 were significantly lower in islets from donors with type 2 diabetes mellitus. To investigate the functional relevance of these clock genes, we correlated their expression to insulin content and glycated hemoglobin levels: mRNA levels of PER2 (ρ = 0.33, P = .012), PER3 (ρ = 0.30, P = .023), and CRY2 (ρ = 0.37, P = .0047) correlated positively with insulin content. Of these genes, expression of PER3 and CRY2 correlated negatively with glycated hemoglobin levels (ρ = -0.44, P = .0012; ρ = -0.28, P = .042). Furthermore, in an in vitro model mimicking pathogenetic conditions, the PER3 mRNA level was reduced in human islets exposed to 16.7 mmol/L glucose per 1 mmol/L palmitate for 48 hours (P = .003). Core clock genes are regulated in human islets. The data suggest that perturbations of circadian clock components may contribute to islet pathophysiology in human type 2 diabetes mellitus.

Circadian clock disruption in the mouse ovary in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Activation of the aryl hydrocarbon receptor (AhR) by the highly toxic, prototypical ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or other dioxin-like compounds compromises ovarian function by altering follicle maturation and steroid synthesis. Although alteration of transcription after nuclear translocation and heterodimerization of AhR with its binding partner, aryl hydrocarbon nuclear transporter (ARNT), is often cited as a primary mechanism for mediating the toxic effects of dioxins, recent evidence indicates that crosstalk between AhR and several other signaling pathways also occurs. Like the circadian clock genes, AhR is a member of the basic helix-loop-helix, Per-ARNT-SIM (bHLH-PAS) domain family of proteins. Thus, these studies tested the hypothesis that TCDD can act to alter circadian clock regulation in the ovary. Adult female c57bl6/J mice entrained to a typical 12h light/12h dark cycle were exposed to a single 1 µg/kg dose of TCDD by gavage. Six days after exposure, animals were released into constant darkness and ovaries were collected every 4h over a 24h period. Quantitative real-time PCR and immunoblot analysis demonstrated that TCDD exposure alters expression of the canonical clock genes, Bmal1 and Per2 in the ovary. AhR transcript and protein, which displayed a circadian pattern of expression in the ovaries of control mice, were also altered after TCDD treatment. Immunohistochemistry studies revealed co-localization of AhR with BMAL1 in various ovarian cell types. Furthermore, co-immunoprecipitation demonstrated time-of-day dependent interactions of AhR with BMAL1 that were enhanced after TCDD treatment. Collectively these studies suggest that crosstalk between classical AhR signaling and the molecular circadian clockworks may be responsible for altered ovarian function after TCDD exposure.

Estimating time of death based on the biological clock.

The biological clock may stop at the time of death in a dead body. Therefore, the biological clock seems useful for estimating the time of death. In this study, we tried to read the biological clock in tissues from dead bodies to estimate the time of death using molecular biological techniques. At first, we examined real-time RT-PCR analysis of gene expression for mPer2 and mBmal1, which constitutes a feedback loop in the oscillation system, in the kidney, liver, and heart of mice. We could detect circadian oscillation of these gene expressions in mouse tissues even at <48 h after death. Thus, the ratio of mPer2/mBmal1 was found to be useful for estimating the time of death. We next applied this method to the liver, kidney, and heart obtained from forensic autopsy cases with less than 72 h of postmortem interval. Significant circadian oscillation of hPer2/hBmal1 ratio could be detected in these autopsy samples. We further examined gene expression for hRev-Erbα, a component of another feedback loop. The ratios of hRev-Erbα/hBmal1 showed higher amplitude of oscillation than those of hPer2/hBmal1 and are considered more suitable for estimating the time of death. In particular, a hRev/hBmal1 ratio of >50 indicated the time of death as 0200-0900 hours, and a hRev/hBmal1 ratio that considerably exceeded 75 indicated the time of death as 0200-0800 hours. On the other hand, a hRev/hBmal1 ratio of less than 25 strongly indicated the time of death as 1000-2300 hours. Taken together, these findings indicate that gene expression analyses of the biological clock could be powerful methods for estimation of the time of death.

Casein kinase 1 delta (CK1delta) regulates period length of the mouse suprachiasmatic circadian clock in vitro.

BACKGROUND: Casein kinase 1 delta (CK1delta) plays a more prominent role in the regulation of circadian cycle length than its homologue casein kinase 1 epsilon (CK1epsilon) in peripheral tissues such as liver and embryonic fibroblasts. Mice lacking CK1delta die shortly after birth, so it has not been possible to assess the impact of loss of CK1delta on behavioral rhythms controlled by the master circadian oscillator in the suprachiasmatic nuclei (SCN). METHODOLOGY/PRINCIPAL FINDINGS: In the present study, mPER2::LUCIFERASE bioluminescence rhythms were monitored from SCN explants collected from neonatal mice. The data demonstrate that SCN explants from neonatal CK1delta-deficient mice oscillate, but with a longer circadian period than littermate controls. The cycle length of rhythms recorded from neonatal SCN explants of CK1epsilon-deficient mice did not differ from control explants. CONCLUSIONS/SIGNIFICANCE: The results indicate that CK1delta plays a more prominent role than CK1epsilon in the maintenance of 24-hour rhythms in the master circadian oscillator.